Bone regeneration using biodegradable polymeric nanocomposite materials and applications of the same

ABSTRACT

A method for producing a biocompatible structure includes: obtaining a load graph and a stress graph representing a relationship between a weight percentage of tissue forming nanoparticles and a maximum load or maximum stress of a polymer film, respectively; determining a first and second weight percentage corresponding to a peak of the load graph and the stress graph respectively; determining an optimal weight percentage based on the first and second weight percentages; preparing a polymer film having the optimal weight percentage of the first tissue forming nanoparticles to the polymer; dividing the polymer film to multiple strips; constructing a scaffold by stacking the strips to form polymer layers and adding bone or composite particles between the polymer layers; applying a solution to the scaffold to form a coated scaffold; and adding second tissue forming particles to the coated scaffold to form the biocompatible structure.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/506,438, filed on Oct. 3, 2014, which itself is a divisional of, andclaims benefit of U.S. patent application Ser. No. 13/947,770(hereinafter, the '770 Application), filed on Jul. 22, 2013, nowallowed. The '770 Application is a continuation-in-part of U.S. patentapplication Ser. No. 11/519,316, filed on Sep. 11, 2006, entitled“SYSTEM AND METHOD FOR TISSUE GENERATION AND BONE REGENERATION” byAlexandru S. Biris and Peder Jensen, now U.S. Pat. No. 8,518,123 anditself claims priority and the benefit of U.S. Provisional ApplicationSer. No. 60/715,841, filed on Sep. 9, 2005, and U.S. ProvisionalApplication Ser. No. 60/726,383, filed on Oct. 13, 2005. The '770Application also claims priority and the benefit of U.S. ProvisionalApplication Ser. No. 61/800,588, filed on Mar. 15, 2013. Thisapplication also relates to co-pending U.S. patent application Ser. No.13/947,827, filed on Jul. 22, 2013, and to co-pending U.S. patentapplication Ser. No. 14/509,719, filed on Oct. 8, 2014, which have thesame inventor and assignee as this application. The entire contents ofthe above identified applications are incorporated herein by reference.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisdisclosure. The citation and/or discussion of such references isprovided merely to clarify the description of the present disclosure andis not an admission that any such reference is “prior art” to thedisclosure described herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference.

STATEMENT OF FEDERALLY-SPONSORED RESEARCH

This disclosure was made with Government support under Grant No.W81XWH-10-2-0130 awarded by the U.S. Department of Defense. TheGovernment has certain rights in the disclosure.

FIELD

The present disclosure relates generally to a biocompatible structurefor bone and tissue regeneration, and more particularly to abiodegradable and bioresorbable nanocomposite incorporating polymer,nanostructured hydroxyapatite and optionally other beneficial factors.

BACKGROUND

The background description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description that may nototherwise qualify as prior art at the time of filing, are neitherexpressly nor impliedly admitted as prior art against the presentdisclosure.

Skeletal deficiencies from trauma, tumors and bone diseases, or abnormaldevelopment frequently require surgical procedures to attempt to restorenormal bone function. Although most of these treatments are successful,they all have problems and limitations.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY

Certain aspects of the present disclosure are directed to a method forproducing a biocompatible structure.

In certain embodiments, the method includes:

-   -   obtaining a load graph representing a functional relationship        between a weight percentage of first tissue forming        nanoparticles in a polymer film and a maximum load of that        polymer film;    -   obtaining a stress graph representing a functional relationship        between the weight percentage of the first tissue forming        nanoparticles in a polymer film and maximum stress of that        polymer film;    -   determining a first weight percentage corresponding to a peak of        the load graph and determining a second weight percentage        corresponding to a peak of the stress graph;    -   determining an optimal weight percentage based on the first and        second weight percentage values;    -   preparing a polymer film having a polymer and the first tissue        forming nanoparticles, wherein a weight percentage of the first        tissue forming nanoparticles to the polymer in the polymer film        is the determined optimal weight percentage;    -   dividing the polymer film into a plurality of strips;    -   constructing a scaffold by stacking the strips to form polymer        layers and adding bone or composite particles between the        polymer layers;    -   applying a second solution to the scaffold to form a coated        scaffold; and adding second tissue forming particles to the        coated scaffold to form the biocompatible structure.

In certain embodiments, the step of preparing the polymer film includes:

-   -   dissolving the polymer in a solvent to form a first solution;    -   adding the first tissue forming nanoparticles to the first        solution to form the second solution wherein a weight percentage        of the first tissue forming nanoparticles to the polymer is the        determined optimal weight percentage; and    -   applying the second solution to a surface to form a polymer film        on the surface, wherein the first tissue forming nanoparticles        are dispersed in the polymer film.

In certain embodiments, the load graph has a first peak and a secondpeak; wherein a weight percentage corresponding to the second peak islarger than a weight percentage corresponding to the first peak; andwherein the first weight percentage is the weight percentagecorresponding to the second peak.

In certain embodiments, the stress graph has a first peak and a secondpeak; wherein a weight percentage corresponding to the second peak islarger than a weight percentage corresponding to the first peak; andwherein the second weight percentage is the weight percentagecorresponding to the second peak.

In certain embodiments, the method further includes:

-   -   determining an upper limit value of a range of the optimal        weight percentage as a maximum value of the first weight        percentage and the second weight percentage plus a first        predetermined percentage;    -   determining a lower limit value of the range as a minimum value        of the first weight percentage and the second weight percentage        minus a second predetermined percentage; and    -   selecting a percentage from the range as the optimal weight        percentage.

In certain embodiments, each of the first and second predeterminedpercentages is about 5%.

In certain embodiments, each of the first and second predeterminedpercentages is about 0%.

In certain embodiments, the method further includes:

-   -   determining an upper limit value of a range of the optimal        weight percentage as an average of the first weight percentage        and the second weight percentage plus a third predetermined        percentage;    -   determining a lower limit value of the range of the optimal        weight percentage as the average minus a fourth predetermined        percentage; and    -   selecting a percentage from the range as the optimal weight        percentage.

In certain embodiments, the third or the fourth predetermined percentageis about 5%.

In certain embodiments, the third or the fourth predetermined percentageis about 0%.

In certain embodiments, the optimal weight percentage is in a range fromabout 0% to about 30%.

In certain embodiments, the optimal weight percentage is about 20%.

In certain embodiments, polymers in the biocompatible polymer filminclude a synthetic biodegradable polymer, a biodegradable polymer fromnatural source, or a mixture thereof. The synthetic biodegradablepolymer includes polyurethane, polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) (PLGA), poly(e-caprolactone), polydioxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH iminocarbonate), poly(bisphenol Aiminocarbonate), poly(ortho ester), polycyanoacrylate, polyphosphazene,or a mixture thereof. The biodegradable polymer derived from naturalsource includes modified polysaccharides, modified proteins, or amixture thereof.

In certain embodiments, the first tissue forming nanoparticles comprisenanoparticles of hydroxypatites, tricalcium phosphates, mixed calciumphosphates and calcium carbonate, bone particles of zenograft, boneparticles of allografts, autografts, bone particles of alloplasticgrafts, or a mixture thereof.

In certain embodiments, the scaffold is formed by stacking the stripsand layers of the second tissue forming particles alternatively.

In certain embodiments, the method further includes plasma treating thebiocompatible structure.

In certain embodiments, the second tissue particles comprises nano-sizedbone particles, micro-sized bone particles, or a mixture thereof.

In certain embodiments, the method further includes adding a thirdtissue forming material to the biocompatible structure. The third tissueforming material comprises a bioactive material, cells, or a mixturethereof. The bioactive material comprises proteins, enzymes, growthfactors, amino acids, bone morphogenic proteins, platelet derived growthfactors, vascular endothelial growth factors, or a mixture thereof. Thecells comprises epithelial cells, neurons, glial cells, astrocytes,podocytes, mammary epithelial cells, islet cells, endothelial cells,mesenchymal cells, stem cells, osteoblast, muscle cells, striated musclecells, fibroblasts, hepatocytes, ligament fibroblasts, tendonfibroblasts, chondrocytes, or a mixture thereof.

Certain aspects of the present disclosure are directed to a method ofproducing a biocompatible structure.

In certain embodiments, the method includes:

-   -   obtaining a first graph representing a functional relationship        between a weight percentage of first tissue forming        nanoparticles in a polymer film and a first property of that        polymer film;    -   obtaining a second graph representing a functional relationship        between the weight percentage of the first tissue forming        nanoparticles in a polymer film and a second property of that        polymer film;    -   determining a first weight percentage corresponding to a peak of        the first graph and determining a second weight percentage        corresponding to a peak of the second graph;    -   determining an optimal weight percentage based on the first and        second weight percentage values;    -   preparing a polymer film having a polymer and the first tissue        forming nanoparticles, wherein a weight percentage of the first        tissue forming nanoparticles to the polymer in the polymer film        is the determined optimal weight percentage;    -   dividing the polymer film into a plurality of strips;    -   constructing a scaffold by stacking the strips to form polymer        layers and adding bone or composite particles between the        polymer layers;    -   applying the second solution to the scaffold to form a coated        scaffold; and    -   adding the second tissue forming particles to the coated        scaffold to form the biocompatible structure.

In certain embodiments, the step of preparing the polymer film includes:

-   -   dissolving the polymer in a solvent to form a first solution;    -   adding the first tissue forming nanoparticles to the first        solution to form a second solution wherein a weight percentage        of the first tissue forming nanoparticles to the polymer is the        determined optimal weight percentage; and    -   applying the second solution to a surface to form a polymer film        on the surface, wherein the first tissue forming nanoparticles        are dispersed in the polymer film.

In certain embodiments, the first graph is a load graph representing afunctional relationship between a weight percentage of tissue formingnanoparticles in a polymer film and a maximum load of that polymer film.The second graph is a stress graph representing a functionalrelationship between the weight percentage of tissue formingnanoparticles in a polymer film and maximum stress of that polymer film.

In certain embodiments, the method further includes determining an upperlimit value and a lower limit value of the optimal weight percentage.The upper limit value is a maximum weight percentage of the first weightpercentage and the second weight percentage plus a first predeterminedpercentage. The lower limit value is a minimum weight percentage of thefirst weight percentage and the second weight percentage minus a secondpredetermined percentage.

In certain embodiments, each of the first and second predeterminedpercentages is about 0%-10%.

In certain embodiments, the optimal weight percentage is chosen from arange of an average of the first weight percentage and the second weightpercentage plus/minus a third predetermined percentage.

In certain embodiments, wherein the third predetermined percentages isabout 0%-10%.

In certain embodiments, the optimal weight percentage of the tissueforming nanoparticles in the polymer is about 20%.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thedisclosure and, together with the written description, serve to explainthe principles of the disclosure. The same reference numbers may be usedthroughout the drawings to refer to the same or like elements in theembodiments.

FIG. 1A illustrates a biocompatible structure according to certainembodiments of the present disclosure;

FIG. 1B illustrates a part of a biocompatible structure according tocertain embodiments of the present disclosure;

FIG. 2 schematically shows a Scanning Electron Microscopy image of abiocompatible structure at a low resolution according to certainembodiments of the present disclosure;

FIGS. 3A-3C schematically show Scanning Electron Microscopy images of abiocompatible structure at a high resolution according to certainembodiments of the present disclosure;

FIG. 4 schematically shows procedures for producing a biocompatiblestructure according to certain embodiments of the present disclosure;

FIGS. 5A and 5B schematically show a pull test set up for measuringmaximum load and maximum stress of polymer films according to certainembodiments of the present disclosure;

FIG. 6 schematically shows maximum load of the polymer films accordingto certain embodiments of the present disclosure; and

FIG. 7 schematically shows maximum stress of the polymer films accordingto certain embodiments of the present disclosure.

DETAILED DESCRIPTION

The present disclosure will now be described more fully hereinafter withreference to the accompanying drawings, in which exemplary embodimentsof the disclosure are shown. This disclosure may, however, be embodiedin many different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the disclosure to those skilled in the art. Likereference numerals refer to like elements throughout. As used in thedescription herein and throughout the claims that follow, the meaning of“a,” “an,” and “the” includes plural reference unless the contextclearly dictates otherwise. Also, as used in the description herein andthroughout the claims that follow, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. Moreover, titles orsubtitles may be used in the specification for the convenience of areader, which has no influence on the scope of the disclosure.Additionally, some terms used in this specification are morespecifically defined below.

Typically, terms such as “first”, “second”, “third”, and the like areused for distinguishing various elements, members, regions, layers, andareas from others. Therefore, the terms such as “first”, “second”,“third”, and the like do not limit the number of the elements, members,regions, layers, areas, or the like. Further, for example, the term“first” can be replaced with the term “second”, “third”, or the like.

Typically, terms such as “about,” “approximately,” “generally,”“substantially,” and the like unless otherwise indicated mean within 20percent, preferably within 10 percent, preferably within 5 percent, andeven more preferably within 3 percent of a given value or range.Numerical quantities given herein are approximate, meaning that the term“about,” “approximately,” “generally,” or “substantially” can beinferred if not expressly stated.

Typically, “nanoscopic-scale,” “nanoscopic,” “nanometer-scale,”“nanoscale,” the “nano-” prefix, and the like refers to elements orarticles having widths or diameters of less than about 1 μm, preferablyless than about 100 nm in some cases. Specified widths can be smallestwidth (i.e. a width as specified where, at that location, the articlecan have a larger width in a different dimension), or largest width(i.e. where, at that location, the article's width is no wider than asspecified, but can have a length that is greater), unless pointed outotherwise.

FIG. 1A schematically shows structure of a biocompatible structure 100according to certain embodiments of the present disclosure. Thebiocompatible structure 100 can be in any shape that conforms to a shapeof an implant site. For example, the biocompatible structure can have acylindrical shape, a rectangular shape, or a spherical shape.

The biocompatible structure includes two or more modified polymer layers102 stacked together. As will be described below, the modified polymerlayers 102 each have nanoparticles 112 dispersed in a polymer matrix114. In certain embodiments, the nanoparticles 112 are hydroxypatite(HAP) nanoparticles. Further, as shown in FIGS. 1A and 1B, spacerparticles 116 are located in between any two of the layers 102 and canfunction as spacer layer 106 between the polymer layers 102. In certainembodiments, the spacer particles 116 each have a diameter of about2-100 μm. In certain embodiments, the spacer particles 116 are partiallyembedded, or trapped, in the surface portion of the polymer layers 102.In certain embodiments, the spacer particles 116 are formed as layers106, and each spacer layer 106 can have a thickness betweenapproximately 0.001 mm and approximately 50 mm, but are typically lessthan 3 mm. The layers can be mechanically stacked or applied in situ ontop of one another. In certain embodiments, the spacer particles 116 canbe bone particles or composite particulates as described below. Incertain embodiments, the spacer particles 116 can be HAP particles asdescribed below. In certain embodiments, a portion of a polymer layer102 can contact a portion of an adjacent polymer layer 102. In certainembodiments, those contacted portions can cross-link with each other. Incertain embodiments, a polymer coating 110 encloses the stacked polymerlayers 102 and spacer layers 106. Further, the surface of the coating110 can have trapped spacer particles 116. In certain embodiments, thespacer particles 116 can form a layer and cover a substantial portion ofthe entire coating 110.

The polymer layers 102 can have different sizes and shapes as desired.In certain embodiments, the polymer layers 102 can be made as strips.For example, the polymer strips 102 each can have a length of 0.005-50cm, a width of 0.002-50 cm, and a thickness of 0.001-50 mm. The size ofthe entire structure 100 can vary in order to match the size of the bonedefect that needs to be regenerated.

In certain embodiments, the polymer matrix 114 of the modified polymerlayer 102 can be polyurethane. The particles 112 dispersed in thepolymer matrix 114 can be hydroxypatite (HAP) nanoparticles. The weightpercentage of the nanoparticles 112 in the polymer film/layer 102 isdefined as the total weight (e.g., grams) of the nanoparticles 112divided by the total of the weight of the nanoparticles 112 (grams) andthe weight of the solid polymers 114 (grams) used for the preparation ofthe polymer film 102. For example, a total of A grams of nanoparticles112 and a total of B grams of polymers 114 are used to manufacture apolymer film 102. The weight percentage of the nanoparticles 112 in thepolymer film 102 is calculated as A/(A+B). In certain embodiments, theweight percentage of HAP nanoparticles 112 in the polymer layer 102 isabout 0.05-95%. In certain embodiments, the weight percentage of HAPnanoparticles 112 in the polymer layer 102 is about 20%.

In certain embodiment, the nanoparticles 112 dispersed in the polymerlayer 102 are Hydroxylapatite nanoparticles and can have a dimensionalrange between 1-100 nanometer (nm). Hydroxylapatite, also calledhydroxyapatite (HA or HAP), is a naturally occurring mineral form ofcalcium apatite with the formula Ca₅(PO₄)₃(OH), but is usually writtenCa₁₀(PO₄)₆(OH)₂to denote that the crystal unit cell comprises twoentities. Hydroxylapatite is the hydroxyl end member of the complexapatite group. The OH⁻ ion can be replaced by fluoride, chloride orcarbonate, producing fluorapatite or chlorapatite. It crystallizes inthe hexagonal crystal system. Pure hydroxylapatite powder is white.Naturally occurring apatites can, however, also have brown, yellow, orgreen colorations, comparable to the discolorations of dental fluorosis.Up to 50% of bone by weight is a modified form of hydroxylapatite (knownas bone mineral). In certain embodiments, the HAP nanoparticlesdispersed in the polymer layer can be composed of pure HAP, havingsignificant crystallinity and very good dispensability due to thepresence of oxygen groups on the surface.

The presence of HAP nanoparticles 112 in the polymer film 114, amongother things, contributes to the pore size and the strength of thepolymer film 114. In addition, the concentration of HAP nanoparticles112 is also related to the degradation rate of the polymer film 114 whenthe polymer film 114 is used as implant material.

In certain embodiments, the HAP nanoparticles 112 can enhancebone/mineralization in bone cells. The HAP nanoparticles 112, togetherwith other nanomaterials, have the ability to increase the osteogenesisand mineralization in bone cells.

In certain embodiment, the spacer particles 116 between the polymerlayers 102 of the present disclosure are bone particles. The boneparticles 116 can be autografts, allografts, xenografts (usually bovine)or alloplastic bone grafts (synthetic, such as tricalcium phosphate). Incertain embodiment, the bone particles 116 are treated with bone mineralproducts, or composite particles. Bones from slaughtered animals are aninexpensive raw material available in large quantities to produce bonemineral. Bones typically contain 50 to 60% of very fine crystallites ofa form of modified hydroxylapatite, which is bonded by collagenic tissueand contains significant qualities of proteinaceous and other matter aswell as associated fat and muscle tissues. Such a modifiedhydroxylapatite, in a pure state and has its essential crystalstructure, represents a highly biocompatible remodeling bone implantmaterial.

In certain embodiments, the bone particles 116 include hydroxyapatitelike crystallites with a particular degree of crystallinity, habit, andsize (irregular platelike morphology, 5-10 nm in thickness 10-50 nm inlength). The specific surface chemistry of the bone particles 116results from the calcium to phosphate ratio (37.5-38.0% calcium and15.5-19.0% phosphorus). The inorganic phase of the bone particle 116contains porosity including ultrastructural interstices (10-100 nm)between the crystallites occurring naturally and produced by removal ofthe organic phase, and microscopic spaces (1-20 μm) including osteocytelacunae, canaliculi, vascular channels, volkman's canals, and the canalsof haversian systems (100-500 nm). The specific surface area, which is ameasure of porosity is in the range 50 to 100 m²/gm as determined bymercury porosimetry. The crystallinity of the bone particle 116 can becharacterized by X-ray diffraction and the porosity and crystallitemorphology and size by electron microscopy.

In certain embodiment, the bone particles 116 of the present disclosureare demineralized bone particles 116 purchased from Geistlich BioOss,INC. The bone particles 116 can be of bovine origin and treated suchthat only the inorganic structure is left, while the organic materialsare removed. The bone particles 116 are composed of powder particleswith a diameter of 0.01-100 micrometer (μm).

In certain embodiments, the spacer particles 116 can be large particlesof HAP that, e.g., are produced in the lab, or composite particles(polymer and inorganic particles).

In certain embodiments, the biocompatible structure 100 can includebioactive materials 126. In certain embodiments, the bioactive materials126 can be sprayed on the surface of the biocompatible structure 100,and/or incorporated in the polymer structures 102 to promote bonegrowth.

The bioactive materials 126 can be proteins/peptides, HA, drugs, growthfactors, antibiotics (such as tetracycline), and bone morphogenicproteins. Preferred bioactive agents 126 are those that enhance tissueregeneration and/or tissue adhesion. Illustrative examples includegrowth factors, antibiotics, immuno-stimulators, andimmuno-suppressants. In one embodiment, the bioactive agent 126 may be abone morphogenic protein such as bone morphogenetic proteins (BMP). Inanother embodiment, the bioactive agent 126 may be a growth factor suchas fibroblast growth factors (FGF) or an agent which promotes thegeneration of connective tissue.

In certain embodiments, tissue can also be grown in vivo by implantingthe biocompatible structure 100 and stem cells or other types ofsuitable cells (liver cells for the growth of liver tissue; myocardialcells, muscle cells for replacing/restoring damaged heart tissue;epithelial cells, connective tissue cells for skin grafts; osteblastsfor bone generation) to an implant site. Alternatively, tissue can begrown in vitro on the biocompatible structure 100 and then implanted(for example, for growth of connective tissue/coronary vessels forarterial grafts).

Suitable living cells can be placed in the biocompatible structurebefore implantation or implanted together with the biocompatiblestructure 100 into a body. The living cells include epithelial cells(e.g., keratinocytes, adipocytes, hepatocytes), neurons, glial cells,astrocytes, podocytes, mammary epithelial cells, islet cells,endothelial cells (e.g., aortic, capillary and vein endothelial cells),and mesenchymal cells (e.g., dermal fibroblasts, mesothelial cells,osteoblasts), smooth muscle cells, striated muscle cells, ligamentfibroblasts, tendon fibroblasts, chondrocytes, fibroblasts, and any of avariety of stem cells. Also suitable for use in the biocompatiblestructure 100,200 are genetically modified cells, immunologically maskedcells, and the like. Appropriate extracellular matrix proteins (ECM) maybe added to the biocompatible structure to further promote cellingrowth, tissue development, and cell differentiation within thescaffold. ECM proteins can include one or more of fibronectin, laminin,vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin,collagen, fibrillin, merosin, anchorin, chondronectin, link protein,bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin,undulin, epiligrin, and kalinin.

Additional bioactive agent 126 incorporated in the biocompatiblestructure 100, among other things, includes biologically activemacromolecules helpful for cell growth, morphogenesis, differentiation,and tissue building, include growth factors, proteoglycans,glycosaminoglycans and polysaccharides. These compounds are believed tocontain biological, physiological, and structural information fordevelopment or regeneration of tissue structure and function.

In certain embodiments, the biocompatible structure 100 can beplasma-treated/activated/electro-sprayed to functionalize the surface ofthe biocompatible structure 100. Surface treatment can improve thehydrophilicity of the biocompatible structure 100 and promote thecolonization of cells and the adhesion of bone particles to the surfaceand pores of the biocompatible structure 100. The surface can also befunctionalized by electron or ion bombardment, laser irradiation and/orby any other physical or chemical surface reaction that affects thebonds near the surface. These processes can also help in sterilizationof the implant. Plasma treatment breaks the surface bonds of thepolymer. After plasma treatment, oxygen atoms “attach” to the surface,changing the surface energy of the surface such that the surface becomesmore hydrophilic and has oxygen and nitrogen rich functional groups.

The biocompatible structure 100 of the present disclosure is highlyporous, biocompatible, and allows for vascular ingrowth for bone/tissueregeneration. The surface typically does not inhibit any biologicalentity from interacting and to be hydrophilic or potentially becomehydrophilic under different conditions or processes. Suitable materialsfor building structures for tissue/bone engineering and regeneration arecertain polymers, ceramics, carbon-based materials and metals and metalcomposites. In certain embodiments, the polymer layers 102 of thebiocompatible structure 100 of the present disclosure are formed frompolyurethane. In certain embodiments, the biocompatible structure 100has a layered structure composed of a polymeric material that maycontain other substances, such as bioactive substances or substancespromoting the generation of tissue growth. Those substances can beformed inside a polymer layer 102 or on the surface of a polymer layer102. Some of the bioresorbable polymers may or may not require enzymesin order to degrade. The layered, porous design gives this structure avery high surface area for neovascularization and the growth of cellsnecessary for tissue regeneration. In addition, stem cells, osteoblasts,and other types of suitable cells can be incorporated into the system toaid in tissue generation. The biocompatible structure 100 can assumedifferent shapes and dimensions as may be required for a particularapplication. The biocompatible structure 100 can be properly positionedin the surgical site directly or with medical pins, screws, or otherdevices.

The biocompatible structure 100 is configured such that the degradationrate or the resorption rate of the biocompatible structure 100 issubstantially matching a rate of tissue generation in the biocompatiblestructure 100. The controllable degradation rate of the biocompatiblestructure 100 can also provide controllable release of the bioactivesubstance or cells formed in the biocompatible structure 100. Thepolymer may have a different degradation rate than that of thebiocompatible structure 100, but it contributes significantly to thedegradation rate of the biocompatible structure 100. Accordingly, apolymer with suitable degradation property is chosen to produce thebiocompatible structure 100 of the present disclosure.

The polymer layers 102 can be degraded by several mechanisms. The mostcommon mechanism is diffusion. Further, the bioactive substances (agent)of the biocompatible structure can diffuse in various manners. Thebioactive agent (drug) can have a core surrounded by an inert diffusionbarrier, which can be membranes, capsules, microcapsules, liposomes, andhollow fibers. Alternatively, the active agent can be dispersed ordissolved in an inert polymer. Drug diffusion through the polymer matrixis the rate-limiting step, and release rates are determined by thechoice of polymer and its consequent effect on the diffusion andpartition coefficient of the drug to be released. By adjusting thediffusion method of the bioactive agent or cells, and components of thebiocompatible structure component, suitable rate of bioactive agent orcells is achieved.

In certain embodiments, after implantation the biocompatible structure100 can be eventually absorbed by the body, for example, by conversionof a material that is insoluble in water into one that iswater/liquid-soluble, and thus need not be removed surgically.

In certain embodiments, the polymer layers 102 in the biocompatiblestructure 100 are biocompatible, processable, sterilizable, and capableof controlled stability or degradation in response to biologicalconditions. The reasons for designing a biocompatible structure 100 thatdegrades over time often go beyond the obvious desire to eliminate theneed for retrieval. For example, the very strength of a rigid metallicimplant used in bone fixation can lead to problems with “stressshielding,” whereas a bioresorbable implant can increase ultimate bonestrength by slowly transferring load to the bone as it heals. For drugdelivery, the specific properties of various degradable systems can beprecisely tailored to achieve optimal release kinetics of the drug oractive agent.

An ideal biodegradable polymer layer 102 for medical applicationstypically has adequate mechanical properties to match the application(strong enough but not too strong), does not induce inflammation orother toxic response, may be fully metabolized once it degrades, and issterilizable and easily processed into a final end product with anacceptable shelf life. In general, polymer degradation is accelerated bygreater hydrophilicity in the backbone or end groups, greater reactivityamong hydrolytic groups in the backbone, less crystallinity, greaterporosity, and smaller finished device size.

A wide range of synthetic biodegradable polymers can be used to form thepolymer matrix 102 of the present disclosure, including polylactide(PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),poly(e-caprolactone), polydioxanone, polyanhydride, trimethylenecarbonate, poly(β-hydroxybutyrate), poly(g-ethyl glutamate), poly(DTHiminocarbonate), poly(bisphenol A iminocarbonate), poly(ortho ester),polycyanoacrylate, and polyphosphazene. There are also a number ofbiodegradable polymers derived from natural sources such as modifiedpolysaccharides (cellulose, chitin, dextran) or modified proteins(fibrin, casein) that can be used to form the polymer matrix of thepresent disclosure.

Other materials can be tyrosine-derived polycarbonate poly(DTE-co-DTcarbonate), in which the pendant group via the tyrosine-an amino acid-iseither an ethyl ester (DTE) or free carboxylate (DT). Through alterationof the ratio of DTE to DT, the material's hydrophobic/hydrophilicbalance and rate of in vivo degradation can be manipulated. It was shownthat, as DT content increases, pore size decreases, the polymers becomemore hydrophilic and anionic, and cells attach more readily.

These materials are subject to both hydrolysis (via ester bonds) andoxidation (via ether bonds). Degradation rate is influenced by PEOmolecular weight and content, and the copolymer with the highest wateruptake degrades most rapidly.

These polymeric materials 102 can also be developed in such a way thatthey are stable in the biological environment, and degrade only underspecific enzymatic conditions (plasmin, etc.). These materials can alsoinclude partially expressed fragments of human or animal fibrin suchthat the system degrades only in contact with plasmin.

The polymer 114 is preferably in solution mixed with a suitable solvent,and other substances can be added to the solution, for example,collagen, drugs, proteins, pep tides, hydroxyapetite crystals (HA), andantibiotics, depending on the type of tissue to be grown. The solutioncan be sonicated to promote mixing of the constituents.

By chosen a suitable polymer 114, the biocompatible structure 100 canachieve controllable supply of therapeutic, analgesic and/orantibacterial substances, growth factors, proteins, peptides, drugs,tissue subcomponents including but not limited to bone particles andhydroxyappetite, which promote growth, prevent infections and the like.

FIG. 2 schematically shows a Scanning Electron Microscopy image of abiocompatible structure 100 at a low resolution according to certainembodiments of the present disclosure. The biocompatible structure 100has bone particles 116 over porous polymer membrane matrix and a hollowinterior to promote cellular growth and blood flow. In FIG. 2, bioactivematerials 126 are shown on the surface of the biocompatible structure100. In certain embodiments, the bioactive materials 126 can be sprayedon the surface of the biocompatible structure 100, and/or incorporatedin the polymer structures 102 to promote bone growth.

FIGS. 3A-3C schematically shows Scanning Electron Microscopy images of abiocompatible structure 100 at high resolutions according to certainembodiments of the present disclosure. As shown in FIGS. 3A-3C, thesurface of the biocompatible structure 100 made from polyurethanepolymer and hydroxyapatite nanoparticles can be very rough and can haveone or more polymeric pores 304. The polymeric pores 304 typically arelarge in size. The size of the polymeric pores 304 can be from about0.001 μm up to about 10 mm. The nanostructural hydroxyapatatite 308 atthe surface of the biocompatible structure 300 can have a size of about1 nm to about 500 nm, and the majority of the nanostructuralhydroxyapatite 308 can have a size of about 2 nm to about 300 nm. Insidethe biocompatible structure 100 is semi-empty due to the spacing betweenthe layers offered by the bone particles. The pore size should vary bothin the range of nanometer (nm) and the range of micrometer (μm).

When placed in an implant site, new tissue of a patient can grow acrossthe pores on the surface of the biocompatible structure, and inside thehollow interior of the biocompatible structure.

In certain embodiments, the biocompatible structure 100 useful for boneand tissue regeneration can be produced by the following procedures: Apolymer 114 is dissolved in a solvent to form a first solution. HAPnanoparticles 112 are added to the first solution to form a secondsolution. The second solution is applied to a surface to form a polymerfilm on the surface. A weight percentage of the first tissue formingmaterial to the polymer is about 0.5-95%. The polymer film is cut into aplurality of strips 102. The biocompatible structure is formed bystacking the strips 102 and placing bone particle layers 106 in betweenthe strips 102. Then the structure is coated by a coating 110 formedfrom the second solution, and bone particles 116 are then added onto thesurface of the coating 110.

(1) Dissolving a Polymer in a Solvent to Form a First Solution.

In certain embodiments, a polymer 114 is dissolved in a solvent to forma first solution. The polymer 114 can be a synthetic biodegradablepolymer, a biodegradable polymer derived from natural source, or theirmixture. In certain embodiment, suitable synthetic biodegradable polymermay include polyurethane, polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) (PLGA), poly(e-caprolactone), polydioxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH iminocarbonate), poly(bisphenol Aiminocarbonate), poly(ortho ester), polycyanoacrylate, polyphosphazene,or their mixture. In certain embodiments, the biodegradable polymerderived from natural source may include modified polysaccharides(cellulose, chitin, dextran), modified proteins (fibrin, casein), ortheir mixture.

In certain embodiment, the polymer 114 is an ester-type hydrophilicpolyurethane with a linear expansion of 50-65%. The water uptake of thepolymer 114 varies with its composition, anywhere from 30-90%. Thepolymer 114 is thermoplastic. Alternatively, a thermosetting polymer 114may work equally well. In certain embodiment, the polymer 114 may bemixed with other polymers to control its degradation rate. In certainembodiment, the polymer is a powder with particles having a diameter ofabout 0.02-50 mm.

The solvent can be methanol or ethanol or any solvent of the polymerused. In certain embodiment, other organic or inorganic solvent (polaraprotic and protic) may also be used. In certain embodiments, thesolvent is at least one of acetone, methyl ethyl ketone, nitromethane,n-propanol, n-butanol, isopropanol, propylene carbonate, dymethilsulfoxide, acetonitrile, dimethylformamide, ethyl acetate, andtetrahydrofuran, dichloromethane.

The polymer 114 is evenly distributed in the first solution. In certainembodiment, low power heating can be used to help the dissolvation ofthe polymer in the solvent. In certain embodiments, stirring is used toaccelerate the uniform distribution of the polymer in the firstsolution. In certain embodiment, after complete dissolvation of thesolid polymer in the solvent, the first solution has a low viscosity.

(2) Adding a First Tissue Forming Material 112 to the First Solution toForm a Second Solution.

The first tissue forming material 112 is then added to the firstsolution to form a second solution. In certain embodiments, the firsttissue forming material 112 may include nanoparticles of hydroxyapatite(HAP), tricalcium phosphates, mixed calcium phosphates and calciumcarbonate, bone particles of zenograft, allografts, autografts,alloplastic grafts, or a mixture thereof.

In certain embodiment, the HAP nanoparticles 112 have a dimensionalrange between 1-100 nm. The HAP nanoparticles 112 can be composed ofpure HAP, having significant crystallinity, and having very gooddispensability due to the presence of oxygen groups on the surface.

The polymer 114 and the first tissue forming material 112 are evenlydistributed in the second solution. In certain embodiments, sonicationis used to accelerate the homogenization of the polymer 114 and thefirst tissue forming material 112 in the second solution.

The weight percentage of the polymer 114 to the first tissue formingmaterial 112 in the second solution is about 20:1 to 2:1. The ratio isrelated with the characteristics of the produced biocompatible structure100. The characteristics of the biocompatible structure 100 includeresistance to load and stress, porosity, degradation rate, etc. Incertain embodiments, the ratio of the polymer 114 to the first tissueforming material 112 can be adjusted to meet requirement of thecondition of a patient, including the bone implant position, size, andmetabolic rate of the patient.

In certain embodiment, the first polymer 114 is polyurethane and thefirst tissue forming material 112 is HAP nanopowder containing HAPnanoparticles. The weight ratio of the added dry HAP nanopowder to thedry mass of the added polymer varies according to the purpose of use.

In certain embodiment, as described below in connection with FIGS. 6-7,if the weight ratio of the dry HAP nanopowders to the dry mass ofpolyurethane is below 25% (i.e., the weight percentage of dry HAPnanopowder in the total weight of dry HAP nanopowder and dry mass ofpolymer is about 20%), the produced polymer film 102 as described belowis strong and hard. If the weight percentage of the dry HAP nanopowdersto the polyurethane is above 40%, the produced the polymer film asdescribed below is weak and breaks easily. In certain embodiment, theHAP nanoparticles 112 do not allow a good crosslinking of the polymerstrands. Therefore the polymer film produced with a high ratio of HAPnanoparticles 112 is very powdery and breaks very easily.

(3) Applying the Second Solution to a Surface to Form a Polymer Film onthe Surface.

In certain embodiment, the polymer film is formed by applying the secondsolution to a surface, and allowing it to dry. In certain embodiment,the second solution can be dried at a room temperature (e.g., 25° C.).In certain embodiment, the second solution is mildly heated to form thepolymer film on the surface, for example, at a temperature higher thanroom temperature (e.g., 25° C.) and lower than 80° C. In certainembodiment, the drying process is under a vacuum condition. In certainembodiment, the surface is a Teflon surface. In certain embodiment, thesurface is a polytetrafluoroethylene (PTFE) surface. In certainembodiment, the second solution can be dried on a PTFE surface undervacuum and under mild heat for less than 24 hours to form the polymerfilm. The thickness of the polymer film can be about 2-10 mm.

(4) Cutting the Polymer Film into a Plurality of Strips.

In certain embodiments, the formed polymer film is cut into theplurality of strips. The strips can be any suitable shape and size toproduce a biocompatible structure with a predetermined shape and size.In certain embodiment, each of the strips 102 is identical to otherstrips. In certain embodiment, each of the strips has a length of about0.002-50 cm, a width of about 0.002-50 cm, and a thickness of about0.001-50 mm.

(5) Forming the Biocompatible Structure 100 by the Strips, the SecondSolution, and a Second Tissue Forming Material.

In certain embodiment, the biocompatible structure 100 is formed fromthe strips, the second solution, and a second tissue forming materialand the following operations:

(a) Constructing a scaffold by stacking the strips to form polymerlayers 102 and adding bone particle layers 106 between the polymerlayers. In certain embodiments, a strip is disposed on a surface as thefirst polymer layer 102. A first layer of bone particles 106 is thenapplied on the first polymer layer 102. A second strip is then used tocover the first bone particle layer 106 to form the second polymer layer102. By alternatively disposing polymer layers 102 and bone particlelayers 106, the scaffold with a predetermined shape and size isconstructed. The scaffold structure composed of polymer layer 102containing HAP nanoparticles 112, bone particle layer 106, polymer layer102 containing HAP nanoparticles 112, bone particle layer 106alternatively. In certain embodiments, at least one polymer layer 102 islocated as one of the outside layers of the scaffold. In certainembodiment, at least one bone particle layer 106 is located as one ofthe outside layers of the scaffold. In order for the entire structure tostay together, methanol or other solvent of the polymer is added by, forexample pipetting, to superficially liquefy the polymer layers 102, suchthat the bone particles 116 can be “trapped” in the polymer layers 102when the structure dries. The bone particles 116 can be partiallyembedded in the polymer layers 102. After the polymer layers 102re-solidifies, the bone particle layers 106 are connected with thepolymer layers 102.

(b) Applying the second solution to the scaffold to form a coatedscaffold. In certain embodiments, the scaffold built as described aboveis then coated by covering with a polymer film that is in a liquid form.In certain embodiment, the second solution is a sticky solution beforeapplying to the scaffold. In certain embodiment, part of the secondsolution poured on the surface of the scaffold penetrates to the insideof the scaffold. The poured second solution forms a coat 110 on thesurface of the scaffold and helps to hold the components of the scaffoldtogether.

(c) In certain embodiment, the forming operation further includes addingthe second tissue forming material to the coated scaffold to form thebiocompatible structure 100. In certain embodiment, the second tissueforming material can be nano-sized bone particles, micro-sized boneparticles, or a mixture thereof. The structure is then allowed to dryovernight under vacuum and mild heat to form the biocompatible structureaccording to the present disclosure.

The biocompatible structure 100 can be any shape and size such that thebiocompatible structure matches the size of the bone defect that needsto be regenerated. In certain embodiment, the biocompatible structurehas a cylindrical shape or a spherical shape. In certain embodiment, thelength of the biocompatible structure is about 2.5 cm (1 inch) and thediameter is about 0.1-1 cm, which matches the diameter of the bone thatneeds to be replaced.

In certain embodiment, the method further includes subjecting thebiocompatible structure 100 to plasma treatment. For example, oncecompletely dried, the biocompatible structure 100 is placed into glassvials for storage. The biocompatible structure 100 is plasma treated bya radio frequency (RF) plasma discharge device, under an environment ofoxygen, nitrogen or a mixture of oxygen and nitrogen. In certainembodiment, the RF plasma treatment time is about 1-3 minutes. Incertain embodiment, the plasma treated biocompatible structure 100 issterilized and sent for animal studies. The purpose of the plasmatreatment is to break the surface bonds of the polymer. After plasmatreatment, oxygen atoms “attach” to the surface, changing the surfaceenergy of the surface such that the surface becomes more hydrophilic andhas oxygen and nitrogen rich functional groups.

In certain embodiment, the method of manufacturing the biocompatiblestructure 100 further includes adding a third tissue forming material tothe biocompatible structure 100. In certain embodiment, the third tissueforming material includes a bioactive material, cells, or a mixturethereof. The bioactive material includes proteins, enzymes, growthfactors, amino acids, bone morphogenic proteins, platelet derived growthfactors, vascular endothelial growth factors, or a mixture thereof. Thecells includes epithelial cells, neurons, glial cells, astrocytes,podocytes, mammary epithelial cells, islet cells, endothelial cells,mesenchymal cells, stem cells, osteoblast, muscle cells, striated musclecells, fibroblasts, hepatocytes, ligament fibroblasts, tendonfibroblasts, chondrocytes, or a mixture thereof.

The biocompatible structure 100 can be any shape, size and weight to fitwith an implant site. In certain embodiment, long bones were surgicallyremoved from the tibia of goats, and biocompatible structures conform tothe implant sited of the goats according to the present disclosure areused for bone regeneration of the goats.

In certain embodiment, when the biocompatible structure 100 is used indental applications for bone generation, the concentration of HAPnanoparticles can be much higher than the concentration of HAPnanoparticles in the biocompatible structure for some other boneregeneration, for example, tibia regeneration. In certain embodiment,the biocompatible structure for dental applications can be crumbled andforms a lot of particles with high surface area.

In certain embodiment, instead of manufacturing the biocompatiblestructure 100 and then using it as implant material, the biocompatiblestructure 100 can also be formed in situ. For example, a first polymerlayer is air sprayed at an implant site or a bone defect area, a firstlayer of bone particles is then added to the polymer layer and depositson the polymer layer. After that, a second polymer layer is air sprayedon the first bone particle layer, followed by adding a second layer ofbone particles. The process is repeated until the biocompatiblestructure, including alternating polymer layers and bone particlelayers, matches the implant site or mimics the bone defect that needs tobe replaced.

In certain embodiment, a Doctor of Medicine (MD) can take a 3D computeraxial tomography scan (CAT) of a patient and sent the result for exampleby emailing the CAT scan file to a manufacturer. The manufacturer thencan build the implant according to the present disclosure to perfectlymatch the actual bone defect.

FIG. 4 illustrates an example of preparing a biocompatible structure 400according to certain embodiments of the present disclosure.

In operation 402, 500 ml methanol is added to a 1 L beaker. The beakeris placed on a magnetic stirrer and a magnetic stir bar is used formixing. 80 grams polyurethane 114 is then added to the methanol in thebeaker. The solution is mixed by the stirring bar to completely dissolvethe polyurethane in the methanol solvent and uniformly distributed thepolyurethane 114 in the solution. The mixing and dissolving ofpolyurethane is at room temperature. In certain embodiment, the solutioncan be heated to accelerate the process.

In operation 406, 20 gram HAP nanoparticles 112 (e.g., Berkeley AdvancedBiomaterials, Inc.) is then added to the solution. Sonication is appliedto guarantee the evenly distribution of the HAP nanoparticles 112 in thesolution.

In operation 410, 10 ml of the solution is pipetted from the beaker andapplied to a PTFE surface. A thin layer of solution is formed on thePTEF surface. The thin layer of solution is allowed to dry at roomtemperature for variable times to form a polymer film. Alternatively,the layer of solution on the PTFE surface can be placed in an oven toheat or low pressure for a period of time to accelerate the formation ofthe polymer film. In certain embodiment, the temperature can be about30-70° C., and the period of time for the heating is about 2-1500minutes. In certain embodiment, the second solution is allowed to dry ona PTFE surface under vacuum under mild heat for less than 24 hours toform the polymer film. The thickness of the polymer film can be about0.01-50 mm.

In operation 414, the polymer film is then cut into identical stripswith a length of about 0.05-20 cm, a width of about 0.02-5 cm, and athickness of about 0.01-50 mm. In certain embodiment, the polymer filmcan be cut into strips with varies shape and size.

In operation 418, a first strip is placed on the PTFE surface to form afirst polymer layer 102. A first layer of bone particles 106 is added onthe surface of the first polymer layer 102. A second strip is placedonto the first bone particle layer 106 to form a second polymer layer102. Then a second bone particle layer 106 is formed on the secondpolymer layer 102. By alternatively disposing the strips and the bondparticle layers, a three-dimensional scaffold is formed with apredetermined shape and size.

In order for the entire structure to stay together, methanol or othersolvent of the polymer is added by, for example pipetting, tosuperficially liquefy the polymer layers 102, such that the boneparticles 116 can be “trapped” in the polymer layers 102 when thestructure dries. The bone particles 116 can be partially embedded in thepolymer layers 102. After the polymer layers 102 re-solidifies, the boneparticle layers 106 are connected with the polymer layers 102.Alternatively, after adding each bone particle layer 106, the methanolor other solvent can be added to trap or embed the bone particles 116 inthe corresponding polymer layers 102.

Next, 1 ml of the methanol/polyurethane/HAP nanoparticle solution isadded to the surface of the three-dimensional scaffold and allowed todry. Accordingly, a coating 110 is formed on the surface of thethree-dimensional scaffold. In certain embodiment, the coating 110 notonly covers the outside of the three-dimensional scaffold, but also canpenetrate to the inside of the three-dimensional scaffold.

Further, bone particles 116 or other suitable particles may be added tothe surface of the coating 110.

In operation 422, the structure is then dried under vacuum overnight. Incertain embodiment, the structure is further subjected to plasmatreatment.

A series of biocompatible structures 100 is produced according to theabove example by varying the HAP concentration. The HAP concentration inthe polymer film is closely related with the characters of the producedbiocompatible structure 100.

FIGS. 5A and 5B show a pull test system 500 used to measure the maximumload and maximum stress of polymer films 550 with various concentrationsof polyurethane and HAP nanoparticle in accordance with certainembodiments of the present disclosure. In one example, the mechanicalbehavior of the composites was analyzed using an ADMET 7600 EXPERTsingle-column, universal, electromechanical testing machine. Theinstrument performs a “pull test” by stretching the polymer film in itsaxial direction and instantaneously produces a “csv” file using the eP2Digital Controller and Gauge Safe Basic Testing Software. The pull testsystem 500 includes a pull test structure 510, a digital controller 530and, optionally, a computer 550. The pull test structure 510 has a base511, a column 513 fixed to and perpendicular to the base 511, a bottomhead 515 connected with two bottom grips 517 a and 517 b facing eachother, a top head 521 connected with two top grips 519 a and 519 bfacing each other, a scale 523 attached to the column 513, and a rail525 placed in the column 513. At least one of the top head 521 and thebottom head 515 is connected with the rail 525 and is movable along therail 525. In this embodiment, the top head 521 is connected through achain or a cable to a motor (not shown) and the chain or the cablepulls/drives the top head 521 along the rail 525. The top grips 519 a/519 b move together and at the same speed with the top head 521.

Polymer films 550 were prepared and tested. In certain embodiment, thepolymer films 550 contain various concentrations of polyurethane and HAPnanoparticles. In one embodiment, the weight percentage of the HAPnanoparticles in the polymer films are 0%, 0.5%, 1%, 2%, 3%, 5%, 10%,15%, 20% and 30% respectively. As described above, the weight percentageof the HAP nanoparticles is defined as the weight of the HAPnanoparticle powder (in gram) used for preparing the polymer filmdivided by the total weight of HAP nanoparticle powder (in gram) andsolid polymers (in gram) used for preparing the polymer film 550. Thepolymer films 550 used in the test have predetermined dimensions. Incertain embodiments, the size of the polymer films 550 is 6 cm×1.5cm×0.02 cm. In certain embodiment, polymer films with the sameconcentration of HAP nanoparticles are prepared with different sizes fortesting.

During the maximum load and maximum stress testing process, the topgrips 521 a/ 521 b and the bottom grips 517 a/ 517 b clip two ends ofthe polymer film 550 in the longtitudial direction of the polymer film550. The dimension of the polymer film 550 and the parameters of theforce to be used are entered into the digital controller 530. In certainembodiments, the length of the polymer film used in the calculation isan effective length, for example, measured by the scale, from the bottomedges of the top grips 519 a/ 519 b to the top edges of the bottom grips517 a/ 517 b. In certain embodiments, if the polymer film 550 clippedbetween the top grips 5191/b and the bottom grips 517 a/b has a dog boneshape, the length used for calculation is the narrow portion of the dogbone shape. When the testing starts, the motor moves at least one of thetop head 521 and the bottom head 515, for example, the top head 521. Thetop grips 519 a/ 519 b move together and at the same speed with the tophead 521 to pull the polymer film 550 at a predetermined speed. Incertain embodiment, the speed can be 0.01-2.5 mm per minute. The topgrips 519 a/ 519 b move along the rail 525 at a predetermined speed topull the polymer film 550 until the polymer film 550 breaks. Theoriginal dimensions of the polymer film 550, the moving speed of the topgrips 519 a/ 519 b, the length of the polymer film 550 immediatelybefore it breaks are recorded. The maximum load and the maximum stressare calculated. In certain embodiments, the calculation is performed bya processor (not shown) in the computer 550. The maximum load is thepull force (newton) applied to the polymer film 550 when the polymerfilm breaks. The maximum stress (KPa) is the pull force applied to thepolymer film 550 when the polymer film 550 breaks divided by thecross-sectional area of the polymer film 550 (the original width timesthe original thickness of the polymer film 550).

The load and stress tests are performed for the polymer films 550 madeaccording to the present disclosure. In certain embodiments, the polymerfilms contain various concentrations of polyurethane and HAPnanoparticles.

FIG. 6 is a load graph of the polymer films 550 in a two dimensionalcoordinate system, which shows a functional relationship between theweight percentage of the HAP nanoparticles in a polymer film and amaximum load of that polymer film. The X-axis of the coordinate systemis the weight percentage of the HAP nanoparticles and the Y-axis of thecoordinate system is the maximum load of the polymer film. As shown inFIG. 6, the maximum load (in newton) for the polymer films 550containing 0%, 0.5%, 1%, 2%, 3%, 5%, 10%, 20% and 30% of HAPnanoparticles are measured and calculated. The maximum load increasessharply from about 20 newton (N) to about 44 N when the HAPconcentration increases from 0% to about 1%. Then the maximum load dropsto about 31 N when the HAP concentration increases from 1% to around10%. After that, the maximum load increases again to about 41 N ataround 20% HAP concentration and drops to about 38 N at around 30% HAPconcentration. Thus, the load graph has two peaks corresponding to 1%and around 20% of HAP concentration. In certain embodiment, the secondpeak at around 20% HAP concentration in the load graph is named loadpeak.

FIG. 7 is a stress graph of the polymer films 550 in a two dimensionalcoordinate system, which shows a functional relationship between theweight percentage of the HAP nanoparticles in a polymer film and amaximum load of that polymer film. The X-axis of the coordinate systemis the weight percentage of the HAP nanoparticles and the Y-axis of thecoordinate system is the maximum stress of the polymer film 550. Asshown in FIG. 7, the maximum stress (in KPa) for the polymer filmscontaining 0%, 0.5%, 1%, 2%, 3%, 5%, 10%, 20% and 30% of HAPnanoparticles are measured and calculated. The maximum stress increasesfrom about 11,000 KPa to about 15,000 KPa when the HAP concentrationincreases from 0% to about 1%. Then the maximum stress decreases toabout 13,600 KPa when the HAP concentration increases from 1% to about3%. After that, the maximum stress increases to about 22,000 KPa whenthe HAP concentration increases from about 3% to about 20%. Furtherincreasing HAP concentration in the polymer films from about 20% to 30%can result in decreasing of the maximum stress from 22,000 to about20,800 KPa. Thus, the stress graph has two peaks corresponding to 1% and20% of HAP concentration. In certain embodiment, the second peak at 20%HAP concentration in the stress graph is named stress peak.

In certain embodiments, a computer 550 can be used to calculate optimalweight percentage of HAP in the polymer film 550 according to the aboveload and stress graphs of a series of polymer films 550. The computer550, utilizing one or more CPUs, can receive the data from the pull teststructure 510 and the digital controller 530, run a calculationsoftware, and then present the result on a monitor.

An optimal weigh percentage of HAP in the polymer film 550 is determinedbased on the results from the load graph and the stress graph by thecomputer 530. In certain embodiments, both the load graph and the stressgraph have at least two peaks. The first peak 604 in the load graphcorresponding to a lower HAP concentration, and the second peak 608 inthe load graph corresponding to a higher HAP concentration. The firstpeak 704 in the stress graph corresponding to a lower HAP concentration,and the second peak 708 in the stress graph corresponding to a higherHAP concentration. The second peak 608 in the load graph is named loadpeak 608, and the second peak 708 in the stress graph is named stresspeak 708. The peak values from the load peak 608 and the stress peak 708are extracted. In this example, both of the load peak 608 and the stresspeak 708 correspond to a HAP weight percentage (HAP concentration) of20%. The maximum value and the minimum value of the load peak 608 andthe stress peak 708 are determined. In this example, both the maximumvalue and the minimum value are 20%. The optimal concentration range hasan upper limit value and a lower limit value. The upper limit value isthe maximum value plus a first predetermined value. The lower limitvalue is the minimum value minus a second predetermined value. Each ofthe first predetermined value and the second predetermined value can be,for example, 10%, 5%, or 0%. Accordingly, in this example, the optimalconcentration range of the HAP in the polymer film is 10%-30%,preferably 15%-25%, and more preferably 20%.

In another example, the load peak 608 and the stress peak 708 havedifferent values. For example, the load peak may be at 17.5% and thestress peak may be at 22.5%. Accordingly, the maximum value is 22.5% andthe minimum value is 17.5%. With the first and second predeterminedvalues at about 10%, preferably 5%, and more preferably 0%, the optimalconcentration ranges of the HAP weight percentage in the polymer filmare 7.5%-32.5%, preferably 12.5%-27.5%, and more preferably 17.5%-22.5%.In other embodiments, the first and second predetermined values can bedifferent values.

In certain embodiments, according to the results shown in FIGS. 6 and 7,the polymer film with 20% HAP concentration shows good structurestability and strength.

In certain embodiments, the biocompatible structure 100 preparedaccording to the present disclosure for the treatment of animals and/orhumans. In certain embodiment, long bones were surgically removed fromthe tibia of goats. For generating long bones of these goats,biocompatible structures of a weight about 1.0-2.5 grams (g) were used.For example, 10 implants with the weight of 2.39 g, 2.34 g, 2.11 g, 1.86g, 2.135 g, 2.18 g, 1.55 g, 2.5 g, 1.22 g, and 1.69 g, respectively,were used to generate long bones for the goats with surgically removedtibia part. For the above 10 examples, the biocompatible structure wasmade by using 4.52 g of polymer (polyurethane), 0.45 g of HAPnanoparticles, and 15 g of bone particles.

The bone growth using the biocompatible structures 100 according toembodiments of the present disclosure has maturity and integrity.

The foregoing description of the exemplary embodiments of the disclosurehas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the disclosure to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments are chosen and described in order to explain theprinciples of the disclosure and their practical application so as toactivate others skilled in the art to utilize the disclosure and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present disclosurepertains without departing from its spirit and scope. Accordingly, thescope of the present disclosure is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

What is claimed is:
 1. A method for producing a biocompatible structure,comprising: obtaining a load graph representing a functionalrelationship between a weight percentage of first tissue formingnanoparticles in a polymer film and a maximum load of that polymer film;obtaining a stress graph representing a functional relationship betweenthe weight percentage of the first tissue forming nanoparticles in apolymer film and maximum stress of that polymer film; determining afirst weight percentage corresponding to a peak of the load graph anddetermining a second weight percentage corresponding to a peak of thestress graph; determining an optimal weight percentage based on thefirst and second weight percentage values; preparing a polymer filmhaving a polymer and the first tissue forming nanoparticles, wherein aweight percentage of the first tissue forming nanoparticles to thepolymer in the polymer film is the determined optimal weight percentage;dividing the polymer film into a plurality of strips; constructing ascaffold by stacking the strips to form polymer layers and adding boneor composite particles between the polymer layers; applying a secondsolution to the scaffold to form a coated scaffold; and adding secondtissue forming particles to the coated scaffold to form thebiocompatible structure.
 2. The method of claim 1, wherein the step ofpreparing the polymer film comprises: dissolving the polymer in asolvent to form a first solution; adding the first tissue formingnanoparticles to the first solution to form the second solution whereina weight percentage of the first tissue forming nanoparticles to thepolymer is the determined optimal weight percentage; and applying thesecond solution to a surface to form a polymer film on the surface,wherein the first tissue forming nanoparticles are dispersed in thepolymer film.
 3. The method of claim 1, wherein the load graph has afirst peak and a second peak; wherein a weight percentage correspondingto the second peak is larger than a weight percentage corresponding tothe first peak; and wherein the first weight percentage is the weightpercentage corresponding to the second peak.
 4. The method of claim 1,wherein the stress graph has a first peak and a second peak; wherein aweight percentage corresponding to the second peak is larger than aweight percentage corresponding to the first peak; and wherein thesecond weight percentage is the weight percentage corresponding to thesecond peak.
 5. The method of claim 1, further comprising: determiningan upper limit value of a range of the optimal weight percentage as amaximum value of the first weight percentage and the second weightpercentage plus a first predetermined percentage; determining a lowerlimit value of the range as a minimum value of the first weightpercentage and the second weight percentage minus a second predeterminedpercentage; and selecting a percentage from the range as the optimalweight percentage.
 6. The method of claim 5, wherein each of the firstand second predetermined percentages is about 5%.
 7. The method of claim5, wherein each of the first and second predetermined percentages isabout 0%.
 8. The method of claim 1, further comprising: determining anupper limit value of a range of the optimal weight percentage as anaverage of the first weight percentage and the second weight percentageplus a third predetermined percentage; determining a lower limit valueof the range of the optimal weight percentage as the average minus afourth predetermined percentage; and selecting a percentage from therange as the optimal weight percentage.
 9. The method of claim 8,wherein the third or the fourth predetermined percentage is about 5%.10. The method of claim 8, wherein the third or the fourth predeterminedpercentage is about 0%.
 11. The method of claim 1, wherein the optimalweight percentage is in a range from about 0% to about 30%.
 12. Themethod of claim 11, wherein the optimal weight percentage is about 20%.13. The method of claim 1, wherein polymers in the biocompatible polymerfilm comprise a synthetic biodegradable polymer, a biodegradable polymerfrom natural source, or a mixture thereof; wherein the syntheticbiodegradable polymer comprises polyurethane, polylactide (PLA),polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),poly(e-caprolactone), polydioxanone, polyanhydride, trimethylenecarbonate, poly(β-hydroxybutyrate), poly(g-ethyl glutamate), poly(DTHiminocarbonate), poly(bisphenol A iminocarbonate), poly(ortho ester),polycyanoacrylate, polyphosphazene, or a mixture thereof; and whereinthe biodegradable polymer derived from natural source comprises modifiedpolysaccharides, modified proteins, or a mixture thereof.
 14. The methodof claim 1, wherein the first tissue forming nanoparticles comprisenanoparticles of hydroxypatites, tricalcium phosphates, mixed calciumphosphates and calcium carbonate, bone particles of zenograft, boneparticles of allografts, autografts, bone particles of alloplasticgrafts, or a mixture thereof.
 15. The method of claim 1, wherein thescaffold is formed by stacking the strips and layers of the secondtissue forming particles alternatively.
 16. The method of claim 1,further comprising plasma treating the biocompatible structure.
 17. Themethod of claim 1, where the second tissue particles comprisesnano-sized bone particles, micro-sized bone particles, or a mixturethereof.
 18. The method of claim 1, further comprising adding a thirdtissue forming material to the biocompatible structure, wherein thethird tissue forming material comprises a bioactive material, cells, ora mixture thereof; wherein the bioactive material comprises proteins,enzymes, growth factors, amino acids, bone morphogenic proteins,platelet derived growth factors, vascular endothelial growth factors, ora mixture thereof; and wherein the cells comprises epithelial cells,neurons, glial cells, astrocytes, podocytes, mammary epithelial cells,islet cells, endothelial cells, mesenchymal cells, stem cells,osteoblast, muscle cells, striated muscle cells, fibroblasts,hepatocytes, ligament fibroblasts, tendon fibroblasts, chondrocytes, ora mixture thereof.
 19. A method for producing a biocompatible structure,comprising: obtaining a first graph representing a functionalrelationship between a weight percentage of first tissue formingnanoparticles in a polymer film and a first property of that polymerfilm; obtaining a second graph representing a functional relationshipbetween the weight percentage of the first tissue forming nanoparticlesin a polymer film and a second property of that polymer film;determining a first weight percentage corresponding to a peak of thefirst graph and determining a second weight percentage corresponding toa peak of the second graph; determining an optimal weight percentagebased on the first and second weight percentage values; preparing apolymer film having a polymer and the first tissue formingnanoparticles, wherein a weight percentage of the first tissue formingnanoparticles to the polymer in the polymer film is the determinedoptimal weight percentage; dividing the polymer film into a plurality ofstrips; constructing a scaffold by stacking the strips to form polymerlayers and adding bone or composite particles between the polymerlayers; applying the second solution to the scaffold to form a coatedscaffold; and adding the second tissue forming particles to the coatedscaffold to form the biocompatible structure.
 20. The method of claim19, wherein the step of preparing the polymer film comprises: dissolvingthe polymer in a solvent to form a first solution; adding the firsttissue forming nanoparticles to the first solution to form a secondsolution wherein a weight percentage of the first tissue formingnanoparticles to the polymer is the determined optimal weightpercentage; and applying the second solution to a surface to form apolymer film on the surface, wherein the first tissue formingnanoparticles are dispersed in the polymer film.
 21. The method of claim19, wherein the first graph is a load graph representing a functionalrelationship between a weight percentage of tissue forming nanoparticlesin a polymer film and a maximum load of that polymer film; and whereinthe second graph is a stress graph representing a functionalrelationship between the weight percentage of tissue formingnanoparticles in a polymer film and maximum stress of that polymer film.22. The method of claim 19, further comprising determining an upperlimit value and a lower limit value of the optimal weight percentage,wherein the upper limit value is a maximum weight percentage of thefirst weight percentage and the second weight percentage plus a firstpredetermined percentage; and wherein the lower limit value is a minimumweight percentage of the first weight percentage and the second weightpercentage minus a second predetermined percentage.
 23. The method ofclaim 22, wherein each of the first and second predetermined percentagesis about 0%-10%.
 24. The method of claim 19, wherein the optimal weightpercentage is chosen from a range of an average of the first weightpercentage and the second weight percentage plus/minus a thirdpredetermined percentage.
 25. The method of claim 24, wherein the thirdpredetermined percentages is about 0%-10%.
 26. The method of claim 19,wherein the optimal weight percentage of the tissue formingnanoparticles in the polymer is about 20%.